iNOS Regulates Diastolic Dysfunction in the Development of Heart Failure

Gen Takagi
Nippon Medical School-Chiba Hokusoh Hospital, Chiba, Japan

Inducible nitric oxide (iNOS) plays a functional role in the development of cardiac decompensation from severely hypertrophied hearts, according to a study presented by Takagi.

The mechanisms by which nitric oxide (NO) regulates the failing heart have been unclear. The lack of understanding may be due to differences in isoform expression and function of NO, that is, iNOS, neuronal NOS, and endothelial NOS may play different roles. Whether iNOS simply plays a role in mediating cytokines, inflammation and vascular function, or whether it exerts an action on the function of the myocyte in heart failure, has not been known.

The present study evaluated the role of iNOS in regulating diastolic dysfunction in the development of left ventricular hypertrophy (LVH) progressing to heart failure (HF) in a canine model. The animals were evaluated in the LVH stage and in the HF stage, and compared to control canines (Figure 1).

The study demonstrated an important role for iNOS with regard to myocyte function in heart failure (Figure 2). iNOS expression (protein level and localization) was significantly enhanced in HF myocytes, and this correlated with diastolic function. Expression of iNOS was not enhanced in LVH or in control animals.

Isolated myocyte length was elongated significantly in LVH and HF compared to controls. Myocyte systolic function and diastolic function were significantly depressed in LVH and HF, compared with controls. L-arginine, a NOS substrate, significantly reduced myocyte function in HF, but not in LVH or controls (Figure 3). Both a specific and a nonspecific iNOS inhibitor abolished this effect of L-arginine in HF myocytes.

Stabilization of Calcium Release Channel (Ryanodine Receptor) Is Involved in the Cardioprotective Mechanism by Angiotensin II Receptor Blocker in Heart Failure

Masafumi Yano
Yamaguchi University, Ube, Japan

The angiotensin II receptor blocker valsartan corrected the abnormal function of the sarcoplasmic reticulum that occurs in heart failure in a study from Yamaguchi University.

Abnormal function of the saroplasmic reticulum (SR) is a major pathogenic mechanism in heart failure. In a canine model of heart failure, valsartan treatment did not improve hemodynamics, but corrected SR function, Yano reported.

In heart failure, an abnormal Ca2+ leak occurs through the ryanodine receptor (RyR). This is due to partial loss of RyR-bound FKBP12.6 and the resulting conformational change in the RyR. The investigators sought to determine whether low-dose valsartan could correct the defective interaction of FKBP12.6 and the RyR in experimental heart failure.

An abnormal SR Ca2+ leak was found in untreated failing SR, but no abnormality was observed in dogs treated with valsartan. Valsartan treatment restored the stoichiometry of the RyR versus FKBP12.6 that was decreased in untreated SR. In the untreated group, the RyR was PKA-hyperphosphorylated, whereas valsartan inhibited PKA hyperphosphorylation of RyR and increased RyR-bound FKBP12.6. The amount of the RyR-bound FKBP12.6 that was tremendously reduced in the untreated group was reversed with valsartan. Both SR Ca2+ uptake function and the amount of Ca2+-ATPase were also decreased in untreated SR, whereas they were restored with valsartan treatment. Valsartan treatment did not improve left ventricular contractility and relaxation at rest, but it did improve the contractile response to dobutamine. Figure 1 illustrates how FKBP-mediated stabilization can interfere and prevent heart failure.

Although valsartan did not improve cardiac function, it corrected SR function. This apparently discordant effect of valsartan may be due to its beta-blockade type action.

Extracellular Collagen Matrix Determines Left Ventricular Shape, Function and Stiffness during the Process of Ventricular Remodeling

Yasuki Kihara
Kyoto University Graduate School of Medicine, Kyoto, Japan

The myocardial cell-to-cell connection through the extracellular collagen matrix (ECM) is important in maintaining left ventricular shape, contraction, and stiffness (Figure 1). ECM may be degraded during the process of left ventricular remodeling (LVR), primarily mediated by the local fibroblasts. The balance between matrix metalloproteinases (MMP) and the tissue inhibitors of MMP (TIMP) appears to be critical for the downstream degradation. Investigators at Kyoto University Graduate School of Medicine found that this ECM breakdown may occur through intrinsic MMPs.

Their study utilized Dahl salt-sensitive rats with hypertension from the age of 11 weeks (compensated, concentric hypertrophy/left ventricular hypertrophy [LVH] stage) until 17 weeks (the LVR stage). The rats were orally administered ONO-4817, a relatively specific MMP-2 inhibitor, or vehicle.

Collagen type I and III were markedly upregulated in the hypertrophy stage, but not in the heart failure stage, in the study rats compared to age-matched normotensive control rats. Zymography showed that both MMP-2 activity and TIMP activity remained inactive in the hypertrophy stage, however the activity of both began to be increased in the heart failure stage. The activation begins at the transcriptional levels.

In the control rats, no changes in MMP activity were found in the hypertrophy stage, but in the remodeling stage net MMP activity increased by 89.2%. This increase was closely related to increases in LV diastolic diameter and systolic wall stress and to a decrease in LV stiffness (Figure 2). This confirms the upregulation of ECM during hypertrophy, which returns towards baseline in heart failure. ECM degradation remained inactive in hypertrophy, but was de novo activated during heart failure transition. Activity of ECM degradation paralleled the LV dilation and mechanical decompensation.

In rats receiving ONO-4817, net MMP-2 activity was suppressed by 27.4% and ECM appeared much denser. In addition, rats receiving ONO-4817 had longer survival than the control rats, about 25-27 weeks compared to 20 weeks for control rats.

In vivo echocardiography showed that in control rats LV diameter increased after 15 weeks, associated with LV wall thinning, decreased LV fractional shortening, and increased LV systolic wall stress, while in the MMP inhibitors groups small LV size, LV shape, systolic wall function, and normal LV wall stress were maintained. Blood pressure levels were not affected by the MMP inhibitor. Therefore, endogenous MMP inhibitor appears to act along with intrinsic MMP activation to provide sufficient suppression of its activation and prevent LV remodeling. Scanning electron microscope showed clear degradation of ECM in the heart failure stage, compared to the hypertrophy stage, while treatment with ONO-4817 showed recovery of the dense collagen network.

In conclusion, an MMP inhibitor, ONO-4817, suppressed the endogenous MMP activation and blocked the ECM degradation that occurred during heart failure transition. The preserved ECM maintained the LV shape, systolic function, and diastolic properties, thus improving animal survival. Pharmacological inhibition of ECM degradation could be an adjuvant therapy for patients undergoing ventricular remodeling.

Extracellular Matrix Remodeling as a Determinant of Transition to Diastolic Heart Failure in Hypertensive Hearts: Its Diagnostic and Therapeutic Approach

Kazuhiro Yamamoto
Osaka University Graduate School of Medicine, Suita, Japan

Extracellular matrix (ECM) remodeling plays crucial roles in diastolic heart failure (DHF), and because of the close relation between fibrosis and brain natriuretic peptide (BNP), the elevation of plasma BNP may be a hallmark of patients with DHF.

Yamamoto and colleagues developed a hypertensive DHF model using Dahl-Iwai salt-sensitive rats to evaluate how myocardial stiffening induces DHF. Myocardial stiffening was not promoted by ventricular hypertrophy (LVH) but by ventricular fibrosis with enhanced collagen cross-link and an increase in type 1, rather than type III, collagen.

Further investigations revealed that BNP may be a useful marker for this structural remodeling. In the DHF model, BNP was associated with maladaptive LVH with progressive ventricular fibrosis, but was not associated with compensatory LVH with subtle fibrosis. This suggested that BNP may be able to discriminate patients at risk for DHF. Indeed, in clinical studies, patients with a history of acute pulmonary edema due to DHF were shown to have higher plasma BNP levels than asymptomatic hypertensive patients with similar ventricular mass, Yamamoto said.

In the DHF model, progression of ventricular fibrosis was associated with phospholipase D (PLD) activation that was induced by growth factors and agonists binding to G-protein-coupled receptors. Since ethanolamine is a product of PLD, the researchers hypothesized that N-methylethanolamine, an analogue of ethanolamine, would decrease PLD activity through a negative feedback mechanism, suppressing collagen production and preventing myocardial stiffening. Indeed this was the case, as this agent suppressed PLD activity and both mRNA and protein levels of collagen without depressor effects, and prevented stiffening. N-methylethanolamine, therefore, may exert therapeutic effects on ventricular fibrosis by inhibiting PLD, independent of stress unloading.

Assessment of Left Ventricular Diastolic Function Independent of Cardiac Translation Using Newly Developed Tissue Strain Imaging with Tissue Tracking Technique

Tomotsugu Tabata
University of Tokushima, Tokushima, Japan

Tissue strain imaging with region-of-interest (ROI) tracking can potentially evaluate left ventricular (LV) diastolic function independent of preload and cardiac translation, according to Tabata, who, with his colleagues, developed the new technique.

LV diastolic function cannot be consistently evaluated by the Doppler transmitral inflow velocity pattern because of its preload dependency. The pulsed tissue Doppler mitral annular motion velocity pattern (TDI) evaluates LV diastolic function believed to be relatively preload-independent, but is limited by the effect of cardiac translation. To overcome the problem of cardiac translation, tissue strain imaging (TSI) was recently developed using color TDI technique (ApliQ, Toshiba Corp.).

With this technique, the center of contraction was set in the LV cavity and velocity was automatically angle-corrected (Figure 1). The velocity values from the same region of moving myocardium were automatically defined and interrogated over time to yield displacement by 2D tissue Doppler tracking technique. TSI was finally obtained as a spatial derivative of the tissue displacement.

A study was performed to evaluate LV diastolic function using variables obtained by TSI with ROI tracking (Figure 2). The study evaluated longitudinal strain rate in 20 normal hearts, 35 hypertrophied hearts and 8 hearts with dilated cardiomyopathy (Figure 3).

The experiments showed that the ratio of early diastolic strain rate obtained from TSI to transmitral E wave velocity consistently decreased from normal to pseudonormal. With this technique, the difference between normal hearts and hypertrophied hearts was highly significant (P <0.0001), as was the difference between normal hearts and those with dilated cardiomyopathy (P <0.0001) (Figure 4). The difference between hypertrophied hearts and those with cardiomyopathy was significant at P <0 .01.

With other techniques, these differences were not revealed. For example, a comparison of the velocity variables by pulsed tissue Doppler showed early diastolic wave to be reduced in hypertrophied hearts and in hearts with cardiomyopathy, versus normal, but the two abnormalities appeared similar and could not be differentiated from each other.

The investigators concluded that the TSI potentially evaluates left ventricular diastolic function in a manner that is free from the problems of preload dependency, cardiac translation, Doppler angle, and tissue tracking.